Dipotassium disulphite

Endpoint summary

Description of key information

skin corrosion: not corrosive, No Category (OECD TG 435; GLP)

skin irritation: skin irritating, Category 2 (OECD TG 439; GLP)

eye irritation/ corrosion: serious eye damaging, Category 1 (OECD TG 405, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-04-20 to 2021-07-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2020-06-26

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2019-02-10

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2019-10-23

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (Standard Assay Kit and MTT-100 kit; MatTek)
- Tissue lot number: 34158, 34179 (for the quality certificate please also refer to the field "Overall remarks, attachments")

TEST FOR MTT INTERFERENCE
- 1 ml of a MTT solution (1 mg/mL) including 50±2 mg of the test item was incubated for 60min (37 ± 1.5°C, 5 ± 0.5% CO2).
- 50 µL deionised water in MTT solution was used as negative control.
- Since the test item was not soluble in isopropanol the suspension was centrifuged.
- After incubation the presence of the staining was evaluated by OD measurement (see section ‎5.5 Measurement).

TEST FOR COLOUR INTERFERENCE
- 50 ± 2 mg of the test item was added to 1 mL of deionised water and mixed. 1 mL of deionised water was used as control (blank). Both were incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2).
- 50 ± 2 mg of the test item was added to 2 mL of isopropanol and mixed. A control (2 mL of isopropanol, blank) was run concurrently. Both were incubated for 60 min at room temperature.
- After incubation the change of colour was determined by the unaided eye.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE AND COLOUR INTERFERENCE
Since the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08 in the first and did not interfere with MTT in the second pre-experiment, no additional tissues were necessary.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min, followed by incubation at room temperature until the 60 min treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1.5 °C

DETAILS OF THE TEST PROCEDURE USED
- Pre-warming:
The inserts with the EpiDerm™ tissues were transferred into 6-well-plates containing 0.9 ml assay medium and incubated for 60 min (37 ± 1.5°C, 5 ± 0.5% CO2). Afterwards, the medium was changed and a further pre-incubation 17 - 23 hours (1st experiment: 21 hours, 20 min; 2nd experiment: 22 hours, 20 min) (37 ± 1.5°C and 5 ± 0.5% CO2).
- Treatment:
The EpiDerm™ tissues were wetted with 25 µL DPBS prior to application. 25 ± 2 mg (39.7 mg/cm2) of the test item and 30 μL of the positive and negative control, respectively, were applied onto the surface of the tissue. Within the exposure time of 60 min the 6-well plates were placed in the incubator for 35 min (37 ± 1.5°C, 5 ± 0.5% CO2) and the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment. At the end of the treatment interval the tissues were gently rinsed with PBS (in-house) several times in order to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
-Post-incubation:
Afterwards, the tissues were incubated in 0.9 mL of fresh assay medium for 24 ± 2 hours (1st experiment: 23 hours, 36 min; 2nd experiment: 24 hours, 15 min). After this incubation period the medium was changed, and the tissues were incubated for another 18 ± 2 hours (1st experiment: 18 hours, 30 min; 2nd experiment: 18 hours, 2 minutes). The complete post-incubation time was 42 ± 4 hours (1st experiment: 42 hours, 6 min; 2nd experiment: 42 hours, 17 min) (37 ± 1.5°C, 5 ± 0.5% CO2).

MTT ASSAY
- For the MTT-assay 24-well plates were prepared with 300 µl MTT (1 mg/mL) solution per well. After post-incubation all tissues were transferred to the prepared MTT-plates. After a 3 h ± 5 minutes incubation period (37 ± 1.5°C, 5 ± 0.5% CO2) the tissues were rinsed with PBS and carefully dried with blotting paper.
- The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted 2-4 hours (1st experiment: 2 hours, 42 min; 2nd experiment: 2 hours, 55 min) while shaking at room temperature.
- Per each tissue, 3 x 200 µL aliquots of the formazan solution were transferred into a 96-well, flat bottom, microtiter plate for OD measurement. 200 µL of isopropanol were added to the wells designated as blanks for 96-well plate.
- Measurement: The optical density (OD570nm) was determined spectrophotometrically in duplicates by a microplate reader (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
The main experiment was performed two-times.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Overall remarks, attachments" below.

PREDICTION MODEL / DECISION CRITERIA
According to OECD TG 439:
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponded to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [(mean ODtest item / positive control) / ODmean of negative control] * 100
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control. The test item is considered to be irritant to skin or serious eye damaging in accordance with Regulation EC No. 1272/2008 (UN GHS “Category 1 or 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. In this case further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification. The test substance may be considered as non-irritant to skin in accordance with Regulation EC No. 1272/2008 (UN GHS “No Category”) if the tissue viability after exposure and post-treatment incubation is more than 50%.

TEST ACCEPTANCE CRITERIA
According to OECD TG 439:
- the mean OD570 of the tissue replicates treated with the negative control should be ≥ 0.8 and ≤ 2.8
- the viability of the tissue replicates treated with the positive control should be ≤ 20%
- the standard deviation calculated from 3 identically treated replicates should be ≤ 18

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the skin irritation potential of the proficiency substances listed in Table 1 of OECD TG 439. The respective proficiency data are attached in the field "Overall remarks, attachments" below.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: The test item was tested neat. Three tissue replicates were wetted with 25 µL of DPBS prior to application and then 25 ± 2 mg (39.7 mg/cm²) of the test item were applied uniformly to the epidermis surface.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

1st experiment: 42 hours, 6 min
2nd experiment: 42 hours, 17 min

Number of replicates:

test item, positive and negative control: in triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

45.62

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

17.25

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
TEST FOR MTT INTERFERENCE
The test item did not reduce MTT to formazan salt after optical evaluation. Therefore, an additional test with freeze-killed tissues was not necessary.

TEST FOR COLOUR INTERFERENCE
The mean OD of the test item in deionised water or isopropanol was < 0.08 and therefore, an additional test with viable tissues without MTT addition was not necessary in the main experiment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of EpiDermTM for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the ten proficiency chemicals listed in Table 1 of OECD TG 439. The respective Laboratory Technical Proficiency Data are attached in the field "Overall remarks, attachments" below.

ACCEPTANCE OF RESULTS:
- the mean OD570 of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 (1.968 and 1.984) in accordance with OECD TG 439.
- the viability of the tissue replicates treated with the positive control is ≤ 20% (3.64% and 4.24%).
- the standard deviation calculated from 3 identically treated replicates is ≤ 18 (0.3 - 4.1).
- Concurrent negative controls and positive controls demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) were within the defined historical acceptance ranges. The results of the positive and negative controls demonstrate reproducibility over time.

Please also refer to the field "Overall remarks, attachments" below.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, in this study and under the experimental conditions reported, Potassium metabisulfite is irritant or corrosive to skin according to UN GHS and EU CLP regulation. Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-10-20 to 2021-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

2015-07-28

Deviations:

yes

Remarks:

minor deviation with no effect on integrity and validity: acc. to the test kit, only single measurements for the positive and negative control instead of quadruplicate. Based on the historical range of breakthrough times, variability in controls is low.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2019-10-23

Test system:

artificial membrane barrier model

Source species:

other: not specified

Cell type:

other: synthetic macromolecular bio-barrier

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

Corrositex TM is a validated and accepted in vitro method to assess if a test item can produce skin corrosion and to distinguish between GHS corrosivity categories 1A, 1B, and 1C.

Vehicle:

unchanged (no vehicle)

Details on test system:

SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Corrositex® test kit: lot no. CT061118; supplier: ROMER Labs Deutschland GmbH, 35510 Butzbach, Germany
- Components: a synthetic macromolecular bio-barrier and a chemical detection system (CDS)
- Apparatus and preparation procedures: One day prior to testing the bio-barrier matrix was prepared. The bio-barrier powder was solved in the bio-barrier diluent and heated for 20 ± 2 minutes at 68 – 70 °C in a water bath under continuous stirring. The temperature did not exceed 70 °C. The mixture was allowed to cool in the turned-off water bath for another 10 minutes. The mixture was then filled into the membrane holders (200 µL per membrane holder). Air bubbles were avoided. The filled membrane holders were sealed with parafilm and were stored at 2 – 8 °C until further use.

WAS THE COMPATIBILITY TEST PERFORMED: yes, the QUALIFY TEST was performed.
In order to test whether the test system is suitable for the test item, 100 mg of the test item were applied into the “Qualify Test Vial”. The vial was shaken until the solution appeared homogenous and incubated for 1 minute. Afterwards, the colour change was noted, and the test proceeded.

WAS THE TIMESCALE CATEGORY TEST PERFORMED: yes, the CATEGORISATION TEST was performed.
In the categorisation test the observation period of the test item after application to the bio-barrier was determined:
- Category 1: observation period after application is 4 hours
- Category 2: observation period after application is 1 hour
Approximately 100 mg of the test item were applied into the “Category A Vial” as well as into the “Category B Vial”. The vials were shaken until the solution appeared homogenous. After 1 minute the colour change was monitored.
No colour change was observed in either of the vials, therefore a confirmation test was conducted. For the confirmation test, two drops of the confirm reagent were added to the “Category B Vial”. The vial was shaken for 5 seconds. After 1 minute the colour change was monitored again. According to the colour scheme the test item was assigned to timescale category 2.

TEMPERATURE USED DURING TREATMENT: room temperature

METHOD OF DETECTION
- Chemical or electrochemical detection system: chemical detection system

METHOD OF APPLICATION / CLASSIFICATION TEST:
Vials containing the CDS were pre-warmed to room temperature. 4 vials were used for quadruplicate measurement of the test item, the vial labelled (+) was used for the positive control Sodium Hydroxide (single measurement). The vial labelled (-) was used for the negative control Citric acid (single measurement). The vial labelled C was used as colour reference for the CDS.
The prepared bio-barriers were placed atop the CDS vials (not longer than 2 min prior to application). Approximately 500 mg of the test item, 500 µl of the negative control Citric acid or 110±15 mg of the positive control Sodium Hydroxide were applied per bio-barrier for 1 hour, depending on the results of the categorisation test. The time interval of the possible colour change or precipitation in the CDS solution was recorded in the raw data file.

PREDICTION MODEL / DECISION CRITERIA:
For each vial the period until observable change in CDS solution was determined. The mean breakthrough time of the quadruplicate measurement was calculated. The test item was categorised according to Table 3 of OECD TG 435.

TEST ACCEPTANCE CRITERIA
According to OECD TG 435:
- the test item induces a physical change (colour or precipitation) in the CDS solution in the qualify test
- the negative control does not induce a physical change (colour or precipitation) up to 60 minutes in the CDS solution in the classification test
- the positive control sodium hydroxide induces a physical change (colour or precipitation) in the CDS solution in the classification test after > 3 – 60 minutes (subcategory 1B)
- the response of the positive control is in the acceptable historical range of breakthrough times for the positive control (mean ± 2-3 standard deviations)

DEMOSTRATING OF PROFICIENCY IN PERFORMING THE TEST METHOD BEFORE ROUTINE USE BY TESTING OF THE PROFICIENCY CHEMICALS
Prior to routine use for regulatory purposes, the laboratory demonstrated technical proficiency by correctly predicting the proficiency substances listed in Table 2 of OECD TG 435. The respective proficiency data are attached in the field "Overall remarks, attachments" below.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 500 mg of the neat test item were dispensed directly atop four bio-barriers.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 500 µL of citric acid (10 % (w/v) solution in deionised water)

POSITIVE CONTROL
- Amount(s) applied (volume or weight):110±15 mg of sodium hydroxide

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

Test item: quadruplicates
Negative control: single measurement
Positive control: single measurement

Irritation / corrosion parameter:

penetration time (in minutes)

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Colour change was not observed up to 60 minutes (treatment period)

Other effects / acceptance of results:

QUALIFY TEST
The test item induced a change in colour in the qualify test after 1 minute incubation. Since a change in colour was visible in the “Qualify Test Vial”, the test item was compatible with the CORROSITEX Assay and the test item was considered to be suitable for the next step.

CATEGORISATION TEST:
Since no colour change was observed in either of the vials, a confirmation test was conducted. For the confirmation test two drops of the confirm reagent were added to the “Category B Vial”. The vial was shaken for 5 seconds and then monitored. The colour of the solution matched one of the CORROSITEXTM colour charts, confirming that the test item was a timescale category 2 substance (observation period 60 minutes).

CLASSIFICATION TEST
A colour change of the CDS reagent after treatment of the bio-barriers with the test item was not observed up to 60 minutes, therefore the test item is non-corrosive to skin.

ACCEPTANCE OF RESULTS:
- The test item induces a physical change in the CDS solution in the qualify test
- No Colour change in the CDS solution of the negative control up to 60 minutes
- The positive control induces a colour change in the CDS solution after 18 minutes (subcategory 1B)
- The exact breakthrough time of the positive control (18 minutes) is in the acceptable historical range of breakthrough times (05:26—28:04) (mean ± 2-3 standard deviations)

Please also refer to the field "Overall remarks, attachments" below.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item is non-corrosive to skin and does not require classification and labelling for skin corrosion according to UN GHS and EU CLP.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Minor deviations with no effect on the results: - Purity and stability of the test substance were no stated - A slightly modified scoring system was used. The naming of the scale of scoring was different, but otherwise the scoring of chemosis/cornea was from 0-4; conjunctivae redness from 0-3 and iris was from 0-2.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

,see "rational for reliability"

GLP compliance:

no

Species:

rabbit

Strain:

Vienna White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: GAUKLER; D-6050 OFFENBACH / MAIN, FRG
- Weight at study initiation: 2.46 kg (male), mean 2.57 kg (females; one female 2.62 kg and the other female 2.51 kg)
- Housing: Individually in cages made of stainless steel with wire mesh walk floors, floor area: 40 x 51 cm; No bedding in the cages; Sawdust in the waste trays.
- Diet: OVATOR SOLIKANIN 4 MM; MUSKATOR-WERKE D-4000 DUESSELDORF-1, FRG (ABOUT 130 G/ANIMAL/DAY )
- Water: About 250 ml tap water/animal/day
- Acclimation period: At least 8 d

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Relative humidity: 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye of same animal

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml bulk volume (about 114 mg of the comminuted test substance) was administered into the conjucntival sac of the right eyelid. The untreated eye served as control.
No further information on the amount/concentration applied was stated.

Duration of treatment / exposure:

one application

Observation period (in vivo):

8 days, Readings at 1, 24, 48, 72 hours and on day 8 after application

Number of animals or in vitro replicates:

1 male / 2 females

Details on study design:

REMOVAL OF TEST SUBSTANCE
The test substance was not washed out.

SCORING SYSTEM:
SCALE FOR SCORING OCULAR LESIONS:
CHEMOSIS (SW) AND CORNEA (OP) (OPACITY-DEGREE OF DENSITY):
O= NONE
1 = SLIGHT
2 = WELL-DEFINED
3 = SEVERE
4 = VERY SEVERE

CONJUNCTIVAE REDNESS (RED):
0 = NORMAL
1 = SLIGHT
2 = WELL-DEFINED
3 = SEVERE

IRIS:
0 = NORMA L
1 = CIRCUM-CORNEAL INJECTION
2 = IRITIS
No further information on the study design was stated.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

not reversible

Remarks:

in 8 days

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Remarks:

in 8 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 8 days

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not reversible

Remarks:

in 8 days

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 8 days

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not fully reversible within: 8 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 8 days

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

not reversible

Remarks:

in 8 days

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Remarks:

in 8 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks:

in 8 days

Irritant / corrosive response data:

During the 1-hour reading slight corneal opacity was seen in one animal. Also, at the 1-hour reading well-defined conjunctival redness and severe chemosis were observed in all animals. Detachment of the cornea and discharge of blood were observed in one male at the 1-hour reading.
At the 24-hour reading suppuration in two animals was observed.
At the 48 hour reading suppuration was observed in all animals.
At the 72 hour reading suppuration was observed in one male and suppuration, small retractions in the eyelids, and pupil contration were observed in one female.
On day 8, the corneal opacitiy worsened in all animals. The conjunctival redness (well-defined) remained the same throughout the observation period, except for one animal in which slight conjunctival redness was observed on day 8.
The chemosis was not fully reversed in any of the animals on day 8, but became better.
On day 8 marginal vascularization of the cornea and small retractions in the eyelids were observed in one female. In addition, marginal vascularization of the cornea was observed in the male rabbit. Furthermore, marginal vascularization of the cornea, small retractions in the eyelids, suppuration, and pupil contration were observed in the remaining female
For raw data see "Any other information on the results incl. tables" below.

Results:

Readings	Animal	cornea		Iris	conjunctiva			Symptons
		opacity	area		redness	swelling	discharge
1 h	1	0	0	0	2	3	2
	2	1	2	0	2	3	2	DC/DB
	3	0	0	0	2	3	2
24 h	1	0	0	0	2	3	2	S
	2	1	2	0	2	2	2	S
	3	0	0	0	2	2	2
48 h	1	1	3	0	2	2	1	S
	2	1	3	0	2	2	2	S
	3	1	2	0	1	1	1	S
72 h	1	1	2	0	2	1	0
	2	1	3	1	2	2	1	S
	3	1	3	0	2	2	1	S/RE/PC
8 h	1	2	2	0	2	1	0	MV/RE
	2	1	2	0	1	1	0	MV
	3	2	3	0	2	2	1	S/RE/PC/MV
mean 24 - 72 h	1	0.7	1.7	0.0	2.0	2.0	1.0
	2	1.0	2.7	0.3	2.0	2.0	1.7
	3	0.7	1.7	0.0	1.7	1.7	1.3
mean		0.8	2.0	0.1	1.9	1.9	1.3

DC - DETACHMENT OF THE CORNEA

DB - DISCHARGE OF BLOOD

MV - MARGINAL VASCULARIZATION OF THE CORNEA

PC - PUPIL CONTRACTED

RE - SMALL RETRACTIONS IN THE EYELIDS

S - SUPPURATION

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is as Category 1 (H319).
Classification: risk of serious damage to eyes

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The substance was observed to be a skin irritant in a reliable in vitro skin irritation test according to OECD TG 439. In a further in vitro test according to OECD TG 435 the substance in non corrosive to skin.

Eye irritation:

The substance was observed to be serious eye damaging in a reliable in vivo eye irritation test equivalent or similar to OECD TG 405.

Justification for classification or non-classification

Skin irritation:

The substance possesses a skin irritation potential based on an in vitro OECD TG 439 test and requires classification as skin irritant (Category 2) according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

The substance possesses a serious eye damaging potential based on an in vivo OECD TG 405 test and requires classification as serious eye damaging (Category 1) according to Regulation (EC) No 1272/2008 and its subsequent adaptations.